This invention relates to the production and purification of polypeptides produced using genetic engineering techniques. More particularly, it relates to genetically engineered fused polypeptides including a novel alpha helical amphiphilic amino acid sequence useful in promoting high expression and in isolating and purifying the fused product.
Advancements in recombinant DNA technology permitting foreign genes to be incorporated into various cells have made possible the expression of products foreign to the cell. The protein of interest will often be degraded by intracellular enzymes, and it may be difficult to separate it from other materials expressed by or comprising the structural materials of the host organism. Protection from intracellular degradation can be achieved by fusing a sequence of amino acids to the target protein to avoid digestion by enzymes within the cell. In addition, the fused protein can be designed to facilitate isolation and purification if the desired protein is fused to a polypeptide having characteristics exploitable in purification.
The fusion product is encoded by DNA comprising the gene coding for the protein of interest linked to a DNA sequence encoding a polypeptide other than the protein of interest. Fusion methodology generally has been discussed at length in the prior art. For example, European Patent Application No. 0047600 is understood to disclose a process for synthesizing bovine growth hormone by producing a fusion protein and purifying the growth hormone from the culture medium of the host organism. Other proteins have been produced through fusion techniques.
Generally, the prior art teaches that genetic material encoding a cleavage site can be incorporated between the DNA encoding the desired protein and the DNA encoding the additional fused material. Expression yields a precursor protein comprising the amino acid sequence of the target polypeptide linked to one or more amino acids defining a selected cleavage site and another amino sequence. See, for example, EPO 0035384, EPO 0161937, and EPO 0163573.
The fusion product can also comprise a moiety designed to facilitate its isolation. For instance, see PCT/84/03103 and U.S. Pat. No. 4,431,739.
It is an object of this invention to provide a method for the production, isolation, and purification of genetically engineered proteins. Another object is to provide a method for obtaining recombinant protein of interest in improved yields. Another object is to provide a method which can be adapted to any polypeptide of interest which can be coded for and expressed by a host organism. Another object is to provide a novel family of leader sequences which induce high levels of expression, which cause the fused polypeptide product it comprises to form insoluble aggregates (inclusion bodies) in the cellular host, and which permit resolubilization and purification of the fused polypeptide. Still another object is to provide such a procedure which is both efficient and inexpensive.
These and other objects of the invention will be apparent from the description, drawing, and claims that follow.